Interspecific Genetic Differences and Historical Demography in South American Arowanas (Osteoglossiformes, Osteoglossidae, Osteoglossum)

The South American arowanas (Osteoglossiformes, Osteoglossidae, Osteoglossum) are emblematic species widely distributed in the Amazon and surrounding basins. Arowana species are under strong anthropogenic pressure as they are extensively exploited for ornamental and food purposes. Until now, limited genetic and cytogenetic information has been available, with only a few studies reporting to their genetic diversity and population structure. In the present study, cytogenetic and DArTseq-derived single nucleotide polymorphism (SNP) data were used to investigate the genetic diversity of the two Osteoglossum species, the silver arowana O. bicirrhosum, and the black arowana O. ferreirai. Both species differ in their 2n (with 2n = 54 and 56 for O. ferreirai and O. bicirrhosum, respectively) and in the composition and distribution of their repetitive DNA content, consistent with their taxonomic status as different species. Our genetic dataset was coupled with contemporary and paleogeographic niche modeling, to develop concurrent demographic models that were tested against each other with a deep learning approach in O. bicirrhosum. Our genetic results reveal that O. bicirrhosum colonized the Tocantins-Araguaia basin from the Amazon basin about one million years ago. In addition, we highlighted a higher genetic diversity of O. bicirrhosum in the Amazon populations in comparison to those from the Tocantins-Araguaia basin. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

Chromosomal Evolution and Evolutionary Relationships of Lebiasina Species (Characiformes, Lebiasinidae)

We present the first cytogenetic data for Lebiasina bimaculata and L. melanoguttata with the aim of (1) investigating evolutionary events within Lebiasina and their relationships with other Lebiasinidae genera and (2) checking the evolutionary relationships between Lebiasinidae and Ctenoluciidae. Both species have a diploid number 2n = 36 with similar karyotypes and microsatellite distribution patterns but present contrasting C-positive heterochromatin and CMA3 + banding patterns. The remarkable interstitial series of C-positive heterochromatin occurring in L. melanoguttata is absent in L. bimaculata. Accordingly, L. bimaculata shows the ribosomal DNA sites as the only GC-rich (CMA3 +) regions, while L. melanoguttata shows evidence of a clear intercalated CMA3 + banding pattern. In addition, the multiple 5S and 18S rDNA sites in L. melanogutatta contrast with single sites present in L. bimaculata. Comparative genomic hybridization (CGH) experiments also revealed a high level of genomic differentiation between both species. A polymorphic state of a conspicuous C-positive, CMA3 +, and (CGG)n band was found only to occur in L. bimaculata females, and its possible relationship with a nascent sex chromosome system is discussed. Whole chromosome painting (WCP) and CGH experiments indicate that the Lebiasina species examined and Boulengerella maculata share similar chromosomal sequences, thus supporting the relatedness between them and the evolutionary relationships between the Lebiasinidae and Ctenoluciidae families

Cytogenetics, genomics and biodiversity of the South American and African Arapaimidae fish family (Teleostei, Osteoglossiformes)

<div><p>Osteoglossiformes represents one of the most ancestral teleost lineages, currently widespread over almost all continents, except for Antarctica. However, data involving advanced molecular cytogenetics or comparative genomics are yet largely limited for this fish group. Therefore, the present investigations focus on the osteoglossiform family Arapaimidae, studying a unique fish model group with advanced molecular cytogenetic genomic tools. The aim is to better explore and clarify certain events and factors that had impact on evolutionary history of this fish group. For that, both South American and African representatives of Arapaimidae, namely <i>Arapaima gigas</i> and <i>Heterotis niloticus</i>, were examined. Both species differed markedly by diploid chromosome numbers, with 2n = 56 found in <i>A</i>. <i>gigas</i> and 2n = 40 exhibited by <i>H</i>. <i>niloticus</i>. Conventional cytogenetics along with fluorescence <i>in situ</i> hybridization revealed some general trends shared by most osteoglossiform species analyzed thus far, such as the presence of only one chromosome pair bearing 18S and 5S rDNA sites and karyotypes dominated by acrocentric chromosomes, resembling thus the patterns of hypothetical ancestral teleost karyotype. Furthermore, the genomes of <i>A</i>. <i>gigas</i> and <i>H</i>. <i>niloticus</i> display remarkable divergence in terms of repetitive DNA content and distribution, as revealed by comparative genomic hybridization (CGH). On the other hand, genomic diversity of single copy sequences studied through principal component analyses (PCA) based on SNP alleles genotyped by the DArT seq procedure demonstrated a very low genetic distance between the South American and African Arapaimidae species; this pattern contrasts sharply with the scenario found in other osteoglossiform species. Underlying evolutionary mechanisms potentially explaining the obtained data have been suggested and discussed.</p></div

Centric fusions behind the karyotype evolution of neotropical nannostomus pencilfishes (Characiforme, Lebiasinidae): First insights from a molecular cytogenetic perspective

Lebiasinidae is a Neotropical freshwater family widely distributed throughout South and Central America. Due to their often very small body size, Lebiasinidae species are cytogenetically challenging and hence largely underexplored. However, the available but limited karyotype data already suggested a high interspecific variability in the diploid chromosome number (2n), which is pronounced in the speciose genus Nannostomus, a popular taxon in ornamental fish trade due to its remarkable body coloration. Aiming to more deeply examine the karyotype diversification in Nannostomus, we combined conventional cytogenetics (Giemsa-staining and C-banding) with the chromosomal mapping of tandemly repeated 5S and 18S rDNA clusters and with interspecific comparative genomic hybridization (CGH) to investigate genomes of four representative Nannostomus species: N. beckfordi, N. eques, N. marginatus, and N. unifasciatus. Our data showed a remarkable variability in 2n, ranging from 2n = 22 in N. unifasciatus (karyotype composed exclusively of metacentrics/submetacentrics) to 2n = 44 in N. beckfordi (karyotype composed entirely of acrocentrics). On the other hand, patterns of 18S and 5S rDNA distribution in the analyzed karyotypes remained rather conservative, with only two 18S and two to four 5S rDNA sites. In view of the mostly unchanged number of chromosome arms (FN = 44) in all but one species (N. eques; FN = 36), and with respect to the current phylogenetic hypothesis, we propose Robertsonian translocations to be a significant contributor to the karyotype differentiation in (at least herein studied) Nannostomus species. Interspecific comparative genome hybridization (CGH) using whole genomic DNAs mapped against the chromosome background of N. beckfordi found a moderate divergence in the repetitive DNA content among the species’ genomes. Collectively, our data suggest that the karyotype differentiation in Nannostomus has been largely driven by major structural rearrangements, accompanied by only low to moderate dynamics of repetitive DNA at the sub-chromosomal level. Possible mechanisms and factors behind the elevated tolerance to such a rate of karyotype change in Nannostomus are discussed. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Cytogenetics of the small-sized fish, Copeina guttata (Characiformes, Lebiasinidae): Novel insights into the karyotype differentiation of the family

Lebiasinidae is a small fish family composed by miniature to small-sized fishes with few cytogenetic data (most of them limited to descriptions of diploid chromosome numbers), thus preventing any evolutionary comparative studies at the chromosomal level. In the present study, we are providing, the first cytogenetic data for the red spotted tetra, Copeina guttata, including the standard karyotype, C-banding, repetitive DNA mapping by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH), providing chromosomal patterns and novel insights into the karyotype differentiation of the family. Males and females share diploid chromosome number 2n = 42 and karyotype composed of 2 metacentric (m), 4 submetacentric (sm) and 36 subtelocentric to acrocentric (st-a) chromosomes. Blocks of constitutive heterochromatin were observed in the centromeric and interstitial regions of several chromosomes, in addition to a remarkably large distal block, heteromorphic in size, which fully corresponded with the 18S rDNA sites in the fourth chromosomal pair. This overlap was confirmed by 5S/18S rDNA dual-color FISH. On the other hand, 5S rDNA clusters were situated in the long and short arms of the 2nd and 15th pairs, respectively. No sex-linked karyotype differences were revealed by male/female CGH experiments. The genomic probes from other two lebiasinid species, Lebiasina melanoguttata and Pyrrhulina brevis, showed positive hybridization signals only in the NOR region in the genome of C. guttata. We demonstrated that karyotype diversification in lebiasinids was accompanied by a series of structural and numeric chromosome rearrangements of different types, including particularly fusions and fissions. © 2019 Toma et al

An Insight into the Chromosomal Evolution of Lebiasinidae (Teleostei, Characiformes)

Lebiasinidae fishes have been historically neglected by cytogenetical studies. Here we present a genomic comparison in eleven Lebiasinidae species, in addition to a review of the ribosomal DNA sequences distribution in this family. With that, we develop ten sets of experiments in order to hybridize the genomic DNA of representative species from the genus Copeina, Copella, Nannostomus, and Pyrrhulina in metaphase plates of Lebiasina melanoguttata. Two major pathways on the chromosomal evolution of these species can be recognized: (i) conservation of 2n = 36 bi-armed chromosomes in Lebiasininae, as a basal condition, and (ii) high numeric and structural chromosomal rearrangements in Pyrrhulininae, with a notable tendency towards acrocentrization. The ribosomal DNA (rDNA) distribution also revealed a marked differentiation during the chromosomal evolution of Lebiasinidae, since both single and multiple sites, in addition to a wide range of chromosomal locations can be found. With some few exceptions, the terminal position of 18S rDNA appears as a common feature in Lebiasinidae-analyzed species. Altogether with Ctenoluciidae, this pattern can be considered a symplesiomorphism for both families. In addition to the specific repetitive DNA content that characterizes the genome of each particular species, Lebiasina also keeps inter-specific repetitive sequences, thus reinforcing its proposed basal condition in Lebiasinidae

Sensitivity of South American tropical forests to an extreme climate anomaly

The tropical forest carbon sink is known to be drought sensitive, but it is unclear which forests are the most vulnerable to extreme events. Forests with hotter and drier baseline conditions may be protected by prior adaptation, or more vulnerable because they operate closer to physiological limits. Here we report that forests in drier South American climates experienced the greatest impacts of the 2015–2016 El Niño, indicating greater vulnerability to extreme temperatures and drought. The long-term, ground-measured tree-by-tree responses of 123 forest plots across tropical South America show that the biomass carbon sink ceased during the event with carbon balance becoming indistinguishable from zero (−0.02 ± 0.37 Mg C ha −1 per year). However, intact tropical South American forests overall were no more sensitive to the extreme 2015–2016 El Niño than to previous less intense events, remaining a key defence against climate change as long as they are protected

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Evolução cromossômica em peixes da família Erythrinidae (Characiformes). Citogenética comparativa entre espécies do gênero Hoplias

The Erythrinidae family is a small group of Neotropical Characiformes, comprising only three genera, Hoplias, Hoplerythrinus and Erythrinus. Hoplias malabaricus, Hoplerythrinus unitaeniatus and Erythrinus erythrinus must correspond to groups of species, considering the extensive chromosomal diversity that they present, including different sex chromosomes systems. Therefore, these fishes offer excellent opportunities for evolutionary investigations, given the different chromosomal characteristics between their representatives. Recent studies allowed the taxonomic characterization of six poorly or not studied Hoplias species (H. aimara, H. lacerdae, H. intermedius, H. brasiliensis, H. australis and H. curupira), present in several South American river basins’ and popularly known as “trairões” due to their large size. This study aimed to characterize the chromosomal evolution occurred in this particular group of species, in the light of its recent review and taxonomic identifications. For this purpose, in addition to classical chromosomal analysis, cytogenetic mapping of repetitive DNA sequences using specific probes was used for fluorescence in situ hybridization (FISH). The results showed the sharing of a great homogeneity in the species´ karyotype macrostructure, with 2n = 50 and karyotypes composed only by meta- and submetacentric chromosomes without the presence of differentiated sex chromosomes between the sexes. In turn, the karyotype microstructure, as revealed by the analysis of components of the repetitive fraction of the genome showed interspecies differentiation, thus confirming the recent taxonomic revision of these species based on their morphological characteristics. This evolutionary scenario, highlighting the conservation of chromosomal macro-structure, sharply contrasts with the scenario found in the representatives of H. malabaricus group, where a conspicuous chromosomal variation can be observed between different populations of this "nominal species." Environmental aspects related to the way of life, as well as intrinsic chromosomal features, may influence the coexistence of these contrasting models of chromosomal evolution among congeneric species of Hoplias.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)A família Erythrinidae é um pequeno grupo de Characiformes Neotropicais, compreendendo apenas três gêneros, Hoplias, Hoplerythrinus e Erythrinus. Hoplias malabaricus, Hoplerythrinus unitaeniatus e Erythrinus erythrinus devem corresponder a grupos de espécies, considerando a ampla diversidade cromossômica que apresentam, incluindo diferentes sistemas de cromossomos sexuais. Assim sendo, os peixes eritrinídeos oferecem excelentes oportunidades para investigações evolutivas, dadas as particularidades diversas entre seus representantes. Estudos recentes possibilitaram a caracterização taxonômica de seis espécies de Hoplias ainda pouco ou não estudadas (H. aimara, H. lacerdae, H. intermedius, H. brasiliensis, H. australis e H. curupira) presentes em diversas bacias hidrográficas do continente sul-americano, dentre as quais se encontram espécimes popularmente conhecidos por trairões devido ao seu grande porte. O presente estudo buscou caracterizar a evolução cromossômica ocorrida nesse grupo particular de espécies, à luz das suas recentes revisões e identificações taxonômicas. Para esta finalidade, além de análises cromossômicas clássicas, foi empregado o mapeamento citogenético de sequências repetitivas de DNA por hibridização fluorescente in situ (FISH). Os resultados mostraram o compartilhamento de uma grande homogeneidade na macroestrutura cariotípica das espécies analisadas, com 2n=50 cromossomos e cariótipos compostos apenas por cromossomos meta- e submetacêntricos, sem a presença de cromossomos sexuais diferenciados entre os sexos. Por sua vez, a microestrutura cariotípica, revelada pela análise de componentes da fração repetitiva do genoma, evidenciou diferenciações interespecíficas, corroborando assim a recente revisão taxonômica dessas espécies com base em suas características morfológicas. Tal cenário evolutivo, destacando a conservação da macro-estrutura cromossômica, contrasta acentuadamente com o cenário presente em representantes do grupo Hoplias malabaricus, onde uma conspícua variação cromossômica pode ser observada entre distintas populações desta “espécie nominal”. Aspectos ecológicos relacionados ao modo de vida, bem como características cromossômicas intrínsecas, podem estar influenciando a coexistência desses modelos contrastantes de evolução cromossômica entre espécies congenéricas de Hoplias

Evolución cromosómica y diversidad genética en especies de Pez de la familia Arapaimidae (Teleostei: Osteoglossiformes)

The Neotropical region exhibits the largest diversity of freshwater fishes in the world and the demographic history of several taxa is related to the geomorphological and climatic history of this region. Among the Osteoglossiformes, the Arapaimidae family is represented by only two genera, with a South American representative Arapaima and another African Heterotis, an important ichthyological group that still has lacked genetic and cytogenetic studies. Therefore, the present work with the use of advanced population genomics tools, selection of demographic models by machine learning, conventional and molecular cytogenetics analysis, aimed at developing a broad understanding of the chromosomal, molecular and phylogeographic diversity of these species. We aimed to better clarify the evolutionary events that shaped the demographic history of this fish group. For that, South American and African representatives of Arapaimidae, namely Arapaima (four natural populations of the Am basin, three of the To-Ar basin and three fish farms) and Heterotis niloticus (a population of the Oluwa River in Nigeria) were examined. The species differed significantly by means of diploid chromosome numbers, with 2n = 56 found in Arapaima and 2n = 40 exhibited by H. niloticus. However, these species presented some general tendencies shared by other Osteoglossiformes analyzed so far, regarding the presence of only one pair of chromosomes bearing 18S and 5S rDNA sites and karyotypes dominated by acrocentric-submetacentric chromosomes, thus corresponding to hypothetical karyotype patterns ancestral to teleosts. In addition, their genomes showed remarkable divergence in terms of repetitive DNA content and distribution as revealed by Comparative Genomic Hybridization (CGH). On the other hand, genomic diversity of single-copy sequences (SNPs), retrieved through principal component analysis (PCA), based on alleles obtained by the DArTseq procedure, demonstrated a very low genetic distance between these genera. These data provided a clear view of the genetic diversity between African and South American Arapaimidae species and were highly consistent with the chromosomal, geographic and historical data, helping to understand its evolutionary diversification. Concerning the investigations of the genus Arapaima in South America, we observed a higher general level of genetic diversity in the Am populations when compared to the one observed in the To-Ar basin. In addition, a high genetic differentiation between the populations of the different basins was observed. By including samples of fish farming, the most likely ancestry of the used breeding herds was revealed, raising concerns about the use of broodstocks outside the distribution of their genetic grouping. No significant chromosome differentiations in the karyotypic structure and repetitive DNA compositions among the studied Arapaima populations was observed. Although the main routes for Arapaima colonization in South America are still unclear, the model selection from convolutional neural networks (CNN) applied to our empirical data indicated a scenario in which the Am basin was the first to be colonized, followed by the To-Ar one. Other studies should focus on a wider historical biogeographic reconstruction of the order Osteoglossiformes, which may potentially explain its relationship with the Gondwana break-up and the presence of this taxon in South America.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)A região Neotropical exibe a maior diversidade de peixes de água doce do mundo e a história demográfica de vários táxons está relacionada com a história geomorfológica e climática desta região. Entre os Osteoglossiformes a família Arapaimidae é representada por apenas dois gêneros, com um representante Sul-Americano Arapaima e outro africano Heterotis, um importante grupo ictiológico que ainda apresenta escassez de dados genéticos. Portanto, as presentes investigações com o uso de ferramentas avançadas da genômica de populações, seleção de modelos demográficos por aprendizado de máquina, citogenética clássica, molecular e citogenômica, objetivaram desenvolver um entendimento amplo da diversidade cromossômica, molecular e filogeográfica destas espécies, com o intuito de esclarecer melhor os eventos evolutivos que moldaram a história demográfica deste grupo de peixes. Para tanto, foram examinados representantes sul-americanos e africanos de Arapaimidae, nomeadamente Arapaima (quatro populações naturais da bacia Am, três da bacia To-Ar e de três pisciculturas) e Heterotis niloticus (uma população do rio Oluwa na Nigéria). Nas abordagens comparativas intercontinentais e intraespecíficas, observou-se que as espécies diferiram significativamente por meio de números cromossômicos diploides, com 2n = 56 encontrado em Arapaima e 2n = 40 exibido por H. niloticus. Entretanto, estas espécies apresentaram algumas tendências gerais compartilhadas por outros osteoglossiformes analisados até o momento, como a presença de apenas um par de cromossomos portadores dos sítios de DNAr 18S e 5S e cariótipos dominados por cromossomos acrocêntricos-submetacêntrico, correspondendo assim aos padrões de cariótipo hipotético ancestral para os teleósteos. Além disso, seus genomas apresentam uma divergência notável em termos de conteúdo e distribuição de DNA repetitivos, como revelado por Hibridização Genômica Comparativa (CGH). Por outro lado, a diversidade genômica de sequências de cópia única (SNPs), estudadas através de análises de componentes principais (PCA), baseadas em alelos obtidos pelo procedimento DArTseq, demonstraram uma distância genética muito baixa entre estes gêneros. Estes dados forneceram uma visão clara da diversidade genética entre espécies africanas e sul-americanas de Arapaimidae e foram altamente consistentes com os dados cromossômicos, geográficos e históricos, auxiliando na compreensão de sua diversificação evolutiva. Já nas investigações do gênero Arapaima para a América do Sul, observamos um nível geral mais alto de diversidade genética nas populações do Am, quando comparado ao observado na bacia To-Ar. Adicionalmente, uma alta diferenciação genética entre as populações das diferentes bacias foi observada. Ao incluirmos amostras de piscicultura, também conseguimos evidenciar a ancestralidade mais provável dos reprodutores usados, levantando a discussão sobre o uso de reprodutores fora da distribuição de seu agrupamento genético. Não houve diferenciações cromossômicas significativas na estrutura cariotípica e nas composições de DNAs repetitivos mapeados entre as populações de Arapaima estudadas. Embora as principais vias para a colonização de Arapaima na América do Sul ainda não estejam claras, a seleção de modelo a partir de redes neurais convolucionais (CNN) aplicadas aos nossos dados empíricos indicam um cenário no qual a bacia do Am foi a primeira a ser colonizada, seguida da bacia To-Ar. Outros estudos devem se concentrar em uma reconstrução biogeográfica histórica mais ampla da ordem Osteoglossiformes, que pode potencialmente explicar sua relação com o rompimento do Gondwana e a presença deste táxon na América do Sul.CAPES: Código de Financiamento 00